Covalent dimeric receptors are found on almost all cells in mammals. These receptors include IR (insulin receptor), IGF-I R (insulin-like growth factor I) and IRR (the insulin receptor-related receptor). In the case of IR, insulin binding to IR is essential for its manifold effects such as glucose homeostasis, increased protein synthesis, growth, and development in mammals. IR belongs to the superfamily of transmembrane receptor TKs that include the monomeric epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR). In contrast, IR and its homologues IGF-I R and IRR are sub-types of this family that are intrinsic disulfide-linked dimers of two heterodimers of the form (αβ)2 (1,2). Monomeric receptor TKs are inactive, but are activated by ligand-induced dimerization that results in autophosphorylation. Dimeric IR-like TKs are also inactive, and are activated by ligand binding without further dimerization. Insulin binding to the extracellular domain of IR results in autophosphorylation of specific tyrosines in the cytoplasmic domain to initiate an intracellular signal transduction cascade (3). However, the structural basis for the mechanism of IR activation by extracellular insulin binding has not been elucidated because the quaternary structure of IR was unknown. Only some of the smaller domains have yielded high resolution structural information.
Diabetes may be caused by mutant IR (eg. acanthosis nigrican or leprechaunism. Insulin resistance leading to diabetes or similar symptoms may also occur.). Diseases are also caused by insufficient amounts of IR ligand. For example, in diabetes, the pancreas produces insufficient amounts of insulin. Insulin activates IR and allows cells to absorb and store glucose. In the absence of adequate insulin, glucose accumulates in excessive amounts in the blood (hyperglycemia). The symptoms of diabetes may include poor blood circulation, blindness and organ damage. These symptoms often lead to premature death.
Diabetes is presently treated by insulin replacement therapy. This treatment has been very successful, but it still has problems such as glycemic control. Poor glycemic control can cause retinopathy, poor blood circulation and the other problems associated with diabetes. It is also difficult to formulate insulin for slow release. Modified insulins have been created in an attempt to address problems with insulin therapy. In some cases, “super-insulins” have been created to increase the activation of insulin receptor by its ligand. In other cases, binding to insulin receptor is not substantially increased, but the ligand has more favourable formulation properties. For example, in Humalog™ (SEQ ID NO:3 and SEQ ID NO:4), a lysine and a proline in insulin are switched to provide more favourable solubility characteristics.
These drug design strategies have been based on limited information, such as the chemical properties of the insulin molecule. In some cases, insulin has been randomly modified and then assayed to determine the effects on insulin activity. While there has been success in producing insulin variants, both of these approaches are time consuming because variants are made without a clear understanding of the effect of the variation on binding to insulin receptor. There is a need to obtain additional information about the insulin receptor in order to provide a rational basis for drug design.
For example, it would be helpful if the quaternary structure, including the ligand binding site, of IR was available and characterized to the detail of amino acids. However, it is very difficult to obtain information about the quaternary structure of dimeric receptors. For example, large transmembrane proteins such as cell surface hormone receptors have been difficult to crystallize as intact molecules for high-resolution structural study. They are also too large for NMR spectroscopy. The 480-kDa insulin receptor (IR) has thus not been crystallized as an intact molecule, and its quaternary structure remains unknown to date.